CN108977429A - A kind of marine microorganism YS2071 stability of lipase protective agent - Google Patents
A kind of marine microorganism YS2071 stability of lipase protective agent Download PDFInfo
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- CN108977429A CN108977429A CN201811071040.XA CN201811071040A CN108977429A CN 108977429 A CN108977429 A CN 108977429A CN 201811071040 A CN201811071040 A CN 201811071040A CN 108977429 A CN108977429 A CN 108977429A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The present invention relates to a kind of marine microorganism YS2071 stability of lipase protective agents; belong to field of biotechnology; the protective agent includes following component: glycerol additive amount 5-10%; sorbierite additive amount 5-15%; fructose additive amount 5-20%, sodium benzoate additive amount 1-2%, calcium chloride additive amount 5-20mM; potassium sorbate additive amount 0.1-1%, most stable pH are 8-10.Protective agent of the present invention substantially increases the stability of marine microorganism YS2071 lipase; it is 91.29% that 90 days enzyme activity retention rates are saved at 25 DEG C; 70.1% is improved than not adding protectant control sample; the stability of marine microorganism YS2071 lipase is greatly improved, is with a wide range of applications.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of marine microorganism YS2071 stability of lipase protection
Agent.
Background technique
An important factor for inactivation of enzyme is limitation enzyme preparation industrial production and application, because the storage stability of enzyme and heat are steady
Qualitative is the key that can enzyme preparation be mass produced and be commercialized, therefore how to improve the storage stability of enzyme and reduce enzyme
Inactivation is the hot spot of biological chemical field research in recent years.The storage stability of enzyme has realistic meaning for the application of enzyme.Mesh
Before, the stabilized main method of enzyme has 3 kinds, i.e. addition protective agent, chemical modification and immobilization processing.Due to method is simple,
The at low cost lower and easily operated most important hand for waiting many-sided reason, adding that protective agent is industrial raising enzyme stability at present
Section.
Lipase is a kind of important hydrolase for acting on acylglycerol, and basic function is that catalyzing glycerol ester hydrolysis is sweet
Oil, diacylglycerol (DGDG), monoglyceride and fatty acid.In addition, lipase can be with catalyzing glycerol ester and water-insoluble esters
The reaction such as hydrolysis, Lipase absobed, acidolysis, transesterification, alcoholysis and ammonolysis, these reactions are usually single-minded with certain alloisomerism
Property and chemo-selective.Compare solid enzyme, and the energy, raw material is greatly saved in liquid enzymes in extraction process, shorten technique,
It reduces costs, reduce environmental pollution, and improve the working condition of worker.However, liquid aliphatic enzyme is in moisture content
Unstable, easy in inactivation in the case where more, acquisition can be such that the protective agent of liquid aliphatic enzyme storage-stable is particularly important.
For marine microorganism because of its unique living environment, the lipase of secretion may have special property and potential use
On the way.Bacterium YS2071 is isolated from Chinese Huanghai Seawater.Studies have shown that the lipase of YS2071 secretion is to long-chain fat
Acid esters has stronger hydrolysis ability, the especially discomposing effect of p-nitrophenyl laurate and laurin preferable,
In addition there is good application potential in terms of catalysis ethyl ester type fish oil is hydrolyzed into fatty acid and in biodiesel conversion.As long-term
Adapt to marine low temperature environment as a result, the thermal stability of the enzyme is poor, just easily inactivate at a lower temperature, this is seriously affected
The development and application of the enzyme.Therefore, the composite protectant of the lipase of invention YS2071 secretion will be established for the application and development of the enzyme
Basis.
Summary of the invention
The technical problem to be solved in the present invention is that providing a kind of marine microorganism YS2071 stability of lipase protective agent.
The present invention is achieved by the following technical solution:
A kind of marine microorganism YS2071 stability of lipase protective agent, the protective agent include the group of following mass fraction
Point: glycerol 5-10%, sorbierite 5-15%, fructose 5-20%, sodium benzoate 1-2%, calcium chloride amount 5-20mM, potassium sorbate
0.1-1%, most stable pH are 8-10；Marine microorganism YS2071 lipase is produced by marine microorganism YS2071.
Another aspect of the invention also provides a kind of marine microorganism YS2071 lipase enzyme solution, is by marine microorganism
YS2071 lipase is added in above-mentioned protective agent and is made, and quality of the marine microorganism YS2071 lipase in protective agent is dense
Degree is 10~15mg/ml.
The above-mentioned protectant preparation method of marine microorganism YS2071 stability of lipase, steps are as follows: in proportion will be sweet
Oil, sorbierite, fructose, sodium benzoate, calcium chloride, potassium sorbate be added 50mM Tris-HCl, pH8.0~10.0 solution in,
It stirs evenly, stands 1~2 hour at 20-30 DEG C, solute is completely dissolved, fixed with the solution of 50mM Tris-HCl, pH8.0~10.0
Hold, marine microorganism YS2071 stability of lipase protective agent is made；
The present invention compared with prior art the utility model has the advantages that
Using stability protection agent provided by the invention, the thermostabilization of marine microorganism YS2071 lipase is substantially increased
Property, enzyme activity retention rate is 91.29% after saving 90 days at 25 DEG C, 70.1% is improved than not adding protectant control sample,
It is with a wide range of applications.
Detailed description of the invention
Fig. 1: influence of the various concentration alcohols to marine microorganism YS2071 lipase activity stability；
Fig. 2: influence of the various concentration carbohydrate to marine microorganism YS2071 lipase activity stability；
Fig. 3: influence of the sodium benzoate to marine microorganism YS2071 lipase activity stability；
Fig. 4: influence of the potassium sorbate to marine microorganism YS2071 lipase activity stability.
Specific embodiment
Below by example to further illustrate the technical scheme of the present invention, but protection scope of the present invention is not appointed by example
The limitation of what form, however it is not limited to following Examples:
Influence of 1 alcohols of embodiment to marine microorganism YS2071 lipase activity stability
Glycerol, the propylene glycol, ethyl alcohol, sorb that isometric content is 1.5,3,4.5,6,7.5,9,10.5% are prepared respectively
Alcoholic solution adds marine microorganism YS2071 Lipase Powder into above-mentioned solution respectively, makes the final concentration of 2mg/mL of enzyme solution, right
Any reagent is not added according to group, each group enzyme solution is placed in 40 DEG C of incubators and places 48h, each group enzyme solution residue enzyme activity is surveyed after 48h.
Polyalcohol (glycerol, propylene glycol, ethylene glycol, polyalcohol-D) etc., polyethylene glycol (PEG), thickener-B, sodium alginate
Deng can and water molecules, reduce the activity of water, zymoprotein made to be influenced to reduce by hydrone, and after these substances are added,
Enzyme solution viscosity increases, and it is more uniform, stable to make enzyme solution system, therefore selection is proper, and the stability of liquid enzymes can be improved.Various concentration
Alcohols to influence such as Fig. 1 of marine microorganism YS2071 lipase activity stability, it can be seen that glycerol and sorbierite are to ocean
Microorganism YS2071 stability of lipase effect is preferable, and highest enzyme activity retention rate is respectively 75.10% and 65.85%, propylene glycol
There is different degrees of inhibiting effect to enzyme activity with ethyl alcohol.
Influence of the different carbohydrates of embodiment 2 to marine microorganism YS2071 stability of lipase
Glucose, alpha-lactose, sugarcane are added respectively into the marine microorganism YS2071 fat enzyme solution of isometric 2mg/mL
Sugar, fructose, xylan, maltose solution, preparing isometric content respectively is respectively 2%, 4%, 6%, 8%, 10%, 12%
Glucose, alpha-lactose, sucrose, fructose, xylan, maltose solution, add enzyme powder respectively into above-mentioned solution, keep enzyme solution whole
Concentration is 2mg/mL, and control group does not add any reagent, and each group enzyme solution is placed in 40 DEG C of incubators and places 48h, is surveyed after 48h each
Group enzyme solution residue enzyme activity.
The carbohydrate of various concentration can therefrom see influence such as Fig. 2 of marine microorganism YS2071 lipase activity stability
Out, fructose has certain protective role to marine microorganism YS2071 lipase activity, and highest enzyme activity retention rate is respectively
65.12%；And other carbohydrates have different degrees of inhibiting effect to enzyme activity.
Influence of 3 preservative of embodiment to marine microorganism YS2071 lipase activity stability
Sodium benzoate and sorbic acid are added respectively into isometric 2mg/ml marine microorganism YS2071 lipase enzyme solution
The preservatives such as potassium make the content 0.4% of additive, and control group does not add any additive, and each group enzyme solution is placed in 40 DEG C of trainings
It supports and places 48h in case, survey each group enzyme solution residue enzyme activity after 48h.
Influence of the sodium benzoate to enzyme activity stability as shown in figure 3, the results show that when sodium benzoate content is 1.6%,
Enzyme activity retention rate reaches highest, is 65.00%, stablizing effect is good.Potassium sorbate to enzyme activity stability influence as shown in figure 4,
When potassium sorbate content is 0.4%, enzyme activity retention rate reaches maximum, is 65.58%, stablizing effect is good.Embodiment 4
A kind of marine microorganism YS2071 stability of lipase protective agent, the protective agent include the group of following mass fraction
Point: glycerol 5%, sorbierite 5%, fructose 5%, sodium benzoate 1%, calcium chloride 5mM, potassium sorbate 0.1%, most stable pH are 8；
The above-mentioned protectant preparation method of marine microorganism YS2071 stability of lipase, steps are as follows: in proportion will be sweet
Oil, sorbierite, fructose, sodium benzoate, calcium chloride, potassium sorbate are added in the solution of 50mM Tris-HCl, pH8.0, and stirring is equal
It is even, 1 hour is stood at 20 DEG C, solute is completely dissolved, and with 50mM Tris-HCl, pH8.0 solution constant volume, marine microorganism is made
The protective agent of YS2071 stability of lipase.
Embodiment 5: a kind of marine microorganism YS2071 stability of lipase protective agent, the protective agent include following quality
The component of score: glycerol 6%, sorbierite 9%, fructose 9%, sodium benzoate 1.7%, calcium chloride 15mM, potassium sorbate 0.5%,
Most stable pH is 9；
The above-mentioned protectant preparation method of marine microorganism YS2071 stability of lipase, steps are as follows: in proportion will be sweet
Oil, sorbierite, fructose, sodium benzoate, calcium chloride, potassium sorbate are added in the solution of 50mM Tris-HCl, pH10.0, stirring
Uniformly, 2 hours are stood at 20-30 DEG C, solute is completely dissolved, and with 50mM Tris-HCl, pH10.0 solution constant volume, ocean is made
The protective agent of microorganism YS2071 stability of lipase；
Embodiment 6
A kind of marine microorganism YS2071 stability of lipase protective agent, the protective agent include the group of following mass fraction
Point: glycerol 8%, sorbierite amount 10%, fructose 15%, sodium benzoate 1.5%, calcium chloride 10mM, potassium sorbate 0.61%, most surely
Determining pH is 9；
The above-mentioned protectant preparation method of marine microorganism YS2071 stability of lipase, steps are as follows: in proportion will be sweet
Oil, sorbierite, fructose, sodium benzoate, calcium chloride, potassium sorbate are added in the solution of 50mM Tris-HCl, pH9.0, and stirring is equal
It is even, 1.5 hours are stood at 25 DEG C, solute is completely dissolved, and with 50mM Tris-HCl, pH9.0 solution constant volume, the micro- life in ocean is made
The protective agent of object YS2071 stability of lipase；
Comparative test
Test 1: marine microorganism YS2071 stability of lipase protective agent+marine microorganism made from embodiment 4
YS2071 lipase；
Test 2: marine microorganism YS2071 stability of lipase protective agent+marine microorganism made from embodiment 5
YS2071 lipase；
Test 3: marine microorganism YS2071 stability of lipase protective agent+marine microorganism made from embodiment 6
YS2071 lipase；
Control group: water+marine microorganism YS2071 lipase
The marine microorganism YS2071 lipase produced is added to the protection of above-described embodiment 4, embodiment 5, embodiment 6
In agent, mass concentration of the lipase in protective agent is 10mg/ml；
Enzyme activity is measured after stablizing and save 90 days at 25 DEG C, test 1, test 2,3 enzyme activity retention rates of test still have 91.29%,
70.1% is improved than not adding protectant control sample.
The measuring method of lipase activity used in the present embodiment: reaction system: A liquid: 83.3mg pNP-lauate is molten
In isopropanol, it is settled to 25mL.B liquid: 0.4%TritonX-100 is added in the phosphate buffer of 0.1mol/L pH 8.0, fixed
Hold to 1L.After 0.1mL A liquid and 1.5mL B liquid are mixed, 4min is preheated in 40 DEG C of thermostat water baths, 0.1mL is then added and waits for
Enzyme solution (control group is the enzyme solution that equivalent boils 8min inactivation) is surveyed, continues to be incubated for 6min, take out, 2mL ethyl alcohol ice bath is added and terminates
Reaction measures light absorption value at 410nm.Enzyme activity unit (U) is defined as under the conditions of 40 DEG C, pH 8.0, and enzyme is catalyzed per minute
Substrate hydrolysis generates the enzyme amount that 1.0 μm of ol/L pNP need.
Claims (3)
1. a kind of marine microorganism YS2071 stability of lipase protective agent, it is characterised in that the protective agent includes such as the following group
Point: glycerol 5-10%, sorbierite 5-15%, fructose 5-20%, sodium benzoate 1-2%, calcium chloride amount 5-20mM, potassium sorbate
0.1-1%, most stable pH are 8-10；Marine microorganism YS2071 lipase is produced by marine microorganism YS2071, above-mentioned hundred
Score is mass fraction.
2. a kind of marine microorganism YS2071 lipase enzyme solution, it is characterised in that the enzyme solution is by marine microorganism YS2071 rouge
Fat enzyme is added in protective agent described in claim 1 and is made, quality of the marine microorganism YS2071 lipase in protective agent
Concentration is 10~15mg/ml.
3. the protectant preparation method of marine microorganism YS2071 stability of lipase described in claim 1, it is characterised in that institute
The step of stating method is as follows: 50mM is added in glycerol, sorbierite, fructose, sodium benzoate, calcium chloride, potassium sorbate in proportion
It in the solution of Tris-HCl, pH8.0~10.0, stirs evenly, stands 1~2 hour at 20-30 DEG C, solute is completely dissolved, and is used
The solution constant volume of 50mM Tris-HCl, pH8.0~10.0.
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| CN201811071040.XA CN108977429B (en) | 2018-09-14 | 2018-09-14 | Marine microorganism YS2071 lipase stability protective agent |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008102264A2 (en) * | 2007-02-20 | 2008-08-28 | Eurand Pharmaceuticals Limited | Stable digestive enzyme compositions |
| CN108497442A (en) * | 2018-04-24 | 2018-09-07 | 东莞市翔实信息科技有限公司 | A kind of cream zymolyte and preparation method thereof |
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2018
- 2018-09-14 CN CN201811071040.XA patent/CN108977429B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008102264A2 (en) * | 2007-02-20 | 2008-08-28 | Eurand Pharmaceuticals Limited | Stable digestive enzyme compositions |
| CN108497442A (en) * | 2018-04-24 | 2018-09-07 | 东莞市翔实信息科技有限公司 | A kind of cream zymolyte and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 姜峻颖: "海洋脂肪酶YS2071的固定化及应用研究", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
| 张权 等: "壳聚糖固定化海洋微生物YS2071脂肪酶及其酶学性质", 《渔业科学进展》 * |
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